Phytochromes (PHYs) are plant photoreceptors essential for regulation of growth and development. There are two main types of PHYs, PHYA and PHYB. PHYB is activated by red (R) and inactivated by far-red (FR) light and has been used in many optogenetic tools. In contrast, PHYA has an action peak at 720 nm (FR) and not at 660 nm (R) even though its photophysical properties are identical to those of PHYB. How exactly plants shift the specificity of PHYA from R to FR light is not understood but it is well established that this is required for survival of plants in canopy shade.

Functional PHYs form homodimers that can exist in three different states: PrPr (both PHYs in the inactive state), PfrPr (one PHY active, one PHY inactive), and PfrPfr (both PHYs active). The relative amount of PfrPfr is highest in R light and drops to nearly zero at ≥700 nm; in contrast, PfrPr levels are highest at 700 nm. Data from stable transgenic plants show that the molecular and kinetic properties of PfrPfr and PfrPr are different and that only PfrPfr is active in case of PHYB, whereas PfrPr is the active species of PHYA.

In the proposed project we want to use a single-molecule pull-down approach to immobilise PHY complexes from plant or mammalian cell lysates and directly visualise their composition and interaction with other proteins by co-localisation of fluorescent markers. Using different PHYA and PHYB mutants we can selectively generate PrPr, PfrPr, or PfrPfr dimers. In addition, we also use stable transgenic plants containing increased levels of PHYA or PHYB PfrPr heterodimers to investigate their physiological activity and molecular properties.